Summary Chronic inflammation in organs with epithelial parenchyma, such as kidney, liver and intestine is a widespread health problem. It is generally accepted that it involves intercellular interactions among epithelial cells and immune cells. It is also known that the epithelial barrier has a role in the maintenance of a chronic inflammation by due increased permeability of tight junctions (TJ) to luminal antigens and bacteria. In turn, TJ are assembled and controlled by signaling pathways involved in apico-basal polarity, such as the Par-polarity complex, which includes Par6, atypical PKC (? and ? isoforms,aPKC), and Par3. Recent work has shown that, in addition, epithelial cells can enhance or limit inflammation by means of pro- or anti-inflammatory cytokines. Using a PKC? conditional knockout mouse model, we have shown that aPKC antagonizes epithelial innate immunity NF-kB and promotes anti-inflammatory IL-10 secretion. Furthermore, we have demonstrated that this mechanism is necessary to prevent a mild pro-inflammatory injury from triggering a deadly acute inflammation. Importantly, under paracrine TNF? stimulation, a small chaperone Bag-1M inhibits normal aPKC refolding and increases its ubiquitination. aPKC chaperoning by Hsp70 occurs on an intermediate filament (keratin) scaffold. Both the keratin-based aPKC refolding and the downstream effects on NF-kB are specific to epithelial cells. The overarching goal of this project is to identify molecular mechanisms, specific to epithelial cells, which result in anti-inflammatory intercellular interactions. The long-term idea is to harness these mechanisms to find novel, alternative therapeutic approaches to chronic inflammation in intestine, liver, or kidney. On the basis of published and preliminary evidence, we hypothesize that, (1) that Bag-1M interacts with keratin-Hsc/Hsp70 complexes at concentrations in the low micromolar range, inhibiting aPKC refolding and subsequent auto-phosphorylation. And (2) aPKC at physiological levels, controls the anti-inflammatory role of the epithelium through indirect inhibition of NF-kB, cytokine transcription, and TJ function. In specific aim 1, we will analyze the mechanisms involved in aPKC destabilization caused by pro-inflammatory signals both paracrine and from microbiota. In specific aim 2 we will study the signaling pathways downstream of aPKC, the relative contribution of TJ permeabilization versus cytokine secretion to local inflammation and possible indirect pathways leading to NF- kB inhibition and control of cytokine transcription by aPKC. We will use a combination of human epithelial cells in culture (Caco-2), non-transformed primary cultures, and conditional PKC?flox/flox crossed with enterocyte or hepatocyte-specific CRE expresser mice to conduct mechanistic and functional experiments to test the hypotheses. All these biological systems have been shown to display the aPKC downregulation upon inflammation, and aPKC-dependent NF-kB inhibition in preliminary data or publications from our lab, thus ensuring the feasibility of all the proposed studies. To our knowledge, we are the first to investigate the role of apico-basal polarity signaling in innate immunity.